Human immunodeficiency virus type 1 (HIV-1) uniquely infects both dividing/activated CD4+ T cell and nondividing cells such as macrophages. These dividing and nondividing HIV-1 target cell types have very different cellular landscapes in terms of the availability of cellular DNA building blocks, dNTPs (macrophages, 20~50nM, and CD4+ T cells, 2-5?M). Recently, our LC-MS/MS study revealed that, unlike dNTPs, macrophages have high rNTP concentrations similar to those of activated T cells. This nondividing cell type thus contains low levels of dNTPs but high levels of rNTPs, unlike activated T cells which contain high levels of both. As a consequence, there is a much larger disparity between the levels of dNTPs vs. rNTPs in macrophages, compared to CD4+ T cells, leading us to hypothesize that HIV-1 may incorporate rNTPs during proviral DNA synthesis in macrophages, but not in activated T cells. Indeed, our recent biochemical simulations revealed that HIV-1 RT uses rNTPs as substrates for DNA synthesis under assay conditions that mimic the dNTP/rNTP pool levels present in macrophages, but not in CD4+ T cell dNTP pool. Furthermore, a ribose-adenosine analog lacking 3' OH inhibited HIV-1 proviral DNA synthesis in macrophages but not in CD4+ T cells, supporting the rNTP incorporation by HIV-1 RT in macrophages. Here, first, we will investigate mechanistic aspects of rNTP and 3' deoxy rNTP incorporation by HIV-1 RT and viral escape from 3' deoxy rNs which will be validated as a new class of macrophage-specific RT chain terminators. Since incorporated rNMPs (ribonucleoside mono-phosphate) in templates are mutagenic, dividing cells harbor a specific repair system to circumvent the mutagenic impact of rNTP incorporation in chromosomes, and RNase H2 and FEN1 are two key enzymes that execute the removal of the singly incorporated rNMPs in ds DNAs. Thus, second, we will test if RNase H2 and FEN1 are expressed in macrophages, and then if the knockdowns of RNase H2 and FEN1 affect HIV-1 infectivity and mutagenesis in macrophages. Lastly, HIV-1 RT has been postulated to undergo a series of conformational changes during dNTP incorporation before chemical catalysis. Recently time-resolved stopped flow florescence technology was employed to monitor the kinetics of this conformational change, and we also recently adopted this technology in our laboratory. In this proposal, third, we will investigate the conformational changes of HIV-1 RT during mutation synthesis events: 1) incorporation of incorrect dNTPs, 2) mismatch extension, 3) rNTP incorporation, and 4) use of mutagenic rNMP containing template by HIV-1 RT and other RT variants. In this renewal application, we will systematically investigate our recent new observations, and continue exploring the mechanistic features of HIV-1 genomic hyper-mutability, and this proposed work is essential for our long-term research goals to develop anti-HIV strategies that can specifically limit the unique viral capability to efficiently mutate and escape during the course of viral pathogenesis and anti-viral treatments.